Since the structure of DNA was deciphered by Watson & Crick in 1953 (Watson, J. D. and Crick, F. H. C., Nature 171 (1953) 737-738 investigation and handling of nucleic acids becomes an integral part of biochemistry molecular biology. Despite the availability of a number of isolation methods an commercial kits for performing such methods, new developments for fast and easy isolation or purification of nucleic acids with high yield and purity are still of major importance.
Nucleic acids are highly susceptible to enzymatic degradation. In 1968 Cox described the chaotropic agent guanidine HCl as an inhibitor of enzymatic nuclease activity (Cox, R. A., Methods Enzymol. 12B (1968) 120-129). Besides a strong denaturing effect on proteins high concentrations of chaotropic agents also mediate cell lysis. Therefore chaotropic agents, particularly guanidine isothiocyanate, are widely in use for nucleic acid isolation.
A first principle of nucleic acid isolation from a biological sample uses an organic solvent, particularly phenol, for the separation of nucleic acids from, the remaining organic sample components. The phenol extraction is followed by a salt precipitation of the nucleic acid from an aqueous phase (Stallcup, M. R. and Washington, L. D., J. Biol. Chem. 258 (1983) 2802-2807, and Schmitt, M. E. et al., Nucl Acid Res 18 (1990) 3091-3092). Although this method results in nucleic acids with high yield and purity the major drawbacks are the use of poisonous reagents, the time consuming and labor intensive workflow. Due to these disadvantages automation of this isolation principle is not amenable to automation, or only to a very limited extent.
Another principle of nucleic acid isolation makes use of solid inorganic material, particularly silica, to which nucleic acids are adsorbed from an aqueous liquid phase such as a lysate of a biological sample. In 1979 Vogelstein and Gillespie described a method for isolating nucleic acid from agarose gel slices by binding nucleic acids to silica particles in presence of highly concentrated sodium iodide (Vogelstein, B. and Gillespie, D., Proc. Natl. Acad. Sci. USA 76 (1979) 615-619).
In addition it was found that the binding of nucleic acids to the solid phase was increased by the addition of anionic or cationic or neutral detergents, in particular TRITON-X100 (Union Carbide Chemicals & Plastics Technology Corporation), sodium dodecyl sulfate, NP40, and TWEEN 20 (ICI Americas Inc.).
Adsorption of a nucleic acid to the solid phase is usually performed in the presence of a potent denaturant such as a chaotropic agent (Boom, R., et al., J. Clin. Microbiol. 28 (1990) 495-503; U.S. Pat. No. 5,234,809). For the isolation process the biological material is mixed with a solution containing the denaturant. The resulting mix is brought into contact with the solid phase material whereby nucleic acid molecules are bound to the surface of the solid phase. Afterwards the solid material is washed with solutions containing decreasing chaotropic salt concentrations and increasing alcohol concentrations, in particular ethanol, in order to further purify the bound nucleic acids from other organic material and contaminating agents. In the last step the solid material is brought into contact with a low salt solution or water under alkaline pH in order to remove the bound nucleic acid from the solid phase. The complete workflow comprises a sample lysis step, a binding step, one or more washing steps, and an elution (desorption) step.
The solid phase can be arranged in different conformations. In a first design the solid phase is in fleece shape and embedded in a plastic device. An example therefor is a micro spin column (EP 0 738 733). This design is preferentially used in workflows which are performed manually. In a second design magnetic silica particles are used as a solid phase (Bartl, K., et al., Clin. Chem. Lab. Med. 36 (1998) 557-559). This design is preferentially used in automated workflows.
A further improvement of this method was observed when aliphatic alcohol (i.e. ethanol or isopropanol) or polyethylene glycol is added to the solution at the binding step (U.S. Pat. No. 6,383,393).
U.S. Pat. No. 6,905,825 discloses addition of organic solvents to the binding buffer. These organic solvents comprise the aliphatic ethers ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol dimethyl ether, propylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, tetrahydrofuran, and 1,4-dioxane, the aliphatic esters propylene glycol monomethyl ether acetate, and ethyl lactate, and the aliphatic ketones hydroxyacetone, acetone, and methyl ethyl ketone.
US 2005/0079535 discloses the use of acetone, acetylacetone, acetonitrile, dimethylsulfoxide, diethylketone, methylethylketone, methylpropylketone, isobutylmethylketone, gamma-butyrolactone, gamma-valerolactone, propylene carbonate, and N-methyl-2-pyrrolidone as well as the use of the cyclic diether dioxane in the binding buffer, in order to adsorb a nucleic acid to a solid phase such as silica.
US 2006/0166368 A1 discloses a liquid solution comprising Tetraethylene glycol dimethyl ether (TED) in a buffer containing (1) a water-miscible organic component such as methanol, ethanol, 1- or 2-propanol, ethylene glycol, propylene glycol, glycerol, acetonitrile, dimethyl sulfoxide, formamide, dimethylformamide, diglyme, triglyme, or tetraglyme, at a concentration of up to 50% (on a volume basis), (2) an acid component such as acetic acid, formic acid, lactic acid, propionic acid, phosphoric acid, trichloroacetic acid, trifluoroacetic acid, citric acid, oxalic acid, or hydrochloric acid, at a concentration of up to 20% (on a volume basis), (3) a buffer such as sodium phosphate, sodium acetate, sodium formate, or sodium citrate, at a pH of from 1 to 6 and a concentration of from 5 to 200 mM, and (4) a detergent such as sodium dodecyl sulfate, TRITON X-100, SB3-10, and TWEEN 20) at a concentration of from 0.005% to 1% (on a weightivolume basis). The liquid solution is used as a solvent of certain dyes which serve as selective labels in protein biochemistry and particularly for methods of protein detection.
The chemical properties of the reagents used in the nucleic acid isolation/purification process determines the quality of the nucleic acid (yield, purity and size) as well as their performance in down-stream workflows, including polymerase or reverse transcriptase based enzymatic reactions (Mullis, K. and Faloona F. A., Methods Enzymol. 155 (1987) 335-350). Furthermore, additional properties of the reagents like toxicity, as well as physical and chemical aspects like flash point and vapor pressure are of major importance.
Recently the analysis of small RNA molecules with 15 to 200 nucleotides gained strong interest. Especially microRNA (miRNA) and small interfering RNA (siRNA), which have a strong effect on the translation of specific messenger RNAs are investigated. Also for other kinds of small RNA like tRNA, 5S and 5.8S rRNA, as well as small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA) involved in mRNA and rRNA processing selective isolation procedures are required.
Methods for isolating such small RNA molecules selectively have been described in US 2005/0059024 by Conrad and in WO 2005/012487 by Madden et al. In order to isolate small RNA molecules in both methods high concentrations of alcohol in the order of 70% is needed to efficiently bind the small RNA to a solid support. This increases the volume of a sample to be analysed considerably. If a sample is to be adsorbed onto a solid support such as a commonly available spin column, the amount of sample that can be applied in one centrifugation run is limited to a small volume. A higher amount of a more diluted sample can be applied only in two consecutive centrifugation steps on the same column, thereby increasing the number of handling steps and processing time. It is therefore another need to improve the binding of small DNA and RNA molecules without the need for diluting the sample with high amounts of alcohol.
In view of the disadvantages of the state of the art it was an object of the present invention to provide an alternative organic compound to promote the adsorption of a nucleic acid to a solid substrate.
The inventors have surprisingly found that adsorption of a nucleic acid to a solid phase is effectively accomplished when tetraethylene glycol dimethyl ether (TDE) is used in the adsorption solution.